G-protein-coupled receptors (GPCRs) are by far the most extensively validated class of therapeutic targets, and there remains tremendous potential for targeting new receptors and their downstream effectors [Neubig et al. Nat Rev Drug Discov, 2002. 1(3): p. 187-97; and Roth et al. Curr Pharm Des, 2006. 12(14): p. 1785-95]. There are over 900 distinct GPCRs encoded in the human genome, and aside from approximately 300 which are involved in odor and taste reception, it is thought that hundreds represent viable targets for therapeutic intervention. Over half of existing drugs are GPCR ligands, yet the total number of receptors that they target is less than 30 [Esbenshade, T. G Protein-Coupled Receptors in Drug Discovery, 2005. Taylor and Francis: p. 15-36]. The proven clinical utility of modulating GPCR signal transduction has sustained formidable efforts in the pharmaceutical industry to identify new GPCR-ligand pairs that control clinically relevant signaling pathways [Mertens et al. Pharmacogenomics, 2004. 5(6): p. 657-72; and Armbruster et al. J Biol Chem, 2004], as well as equally vigorous efforts to discover further components of the GPCR signaling machinery that have the potential to become therapeutic targets.
GPCRs are extensively involved in signal transduction in the nervous system, serving as receptors for the major classes of neurotransmitters including GABA, dopamine and serotonin. Most of the drugs used to treat neurological disorders, including pain relievers, antidepressants and anti-psychotics, exert their effects through GPCRs [Esbenshade, T. G Protein-Coupled Receptors in Drug Discovery, 2005. Taylor and Francis: p. 15-36]. These include dopamine and dopaminergic agents for the treatment of Parkinson's disease and cholinergic agents for the treatment of Alzheimer's disease. Of the 2.2 billion prescriptions issued for the top 200 drugs in 2003, 527 million were for drugs targeting GPCRs, and 147 million were for pain medications targeting an opioid receptor alone—more than the total prescriptions for any other single target class [Esbenshade, T. G Protein-Coupled Receptors in Drug Discovery, 2005. Taylor and Francis: p. 15-36]. Given their extensive role in neurotransmission, GPCR signal transduction pathways clearly represent promising targets for improving the treatment of neurodegenerative diseases. Moreover, development of strategies for modulating these pathways more selectively would expand the potential for more effective treatments.
The standard model of GPCR signal transduction had long been restricted to a three-component system: receptor, G protein and effector [Gilman, A. G. Annu Rev Biochem, 1987. 56: p. 615-49]. The receptor, a cell-surface protein that spans the membrane seven times, is coupled to a membrane-associated heterotrimeric complex that comprises a GTP-hydrolyzing Gα subunit and a Gβγ dimeric partner. Agonist-induced conformational changes enhance the guanine-nucleotide-exchange activity of the receptor, leading to the release of GDP (and subsequent binding of GTP) by the Gα subunit depicted in FIG. 1. On binding GTP, conformational changes within the three ‘switch’ regions of Gα allow the release of Gβγ. Separated Gα·GTP and GPβγ subunits are thus free to propagate signaling forward via separate (and sometimes converging) interactions with adenylyl cyclases, phospholipase-C (PLC) isoforms, potassium and calcium ion channels, guanine-nucleotide exchange factors for the small GTPase RhoA, and other effector systems (FIG. 1). The intrinsic GTP hydrolysis (GTPase) activity of Gα resets the cycle by forming Gα·GDP, which has low affinity for effectors but high affinity for Gβγ. In this way, the inactive, GDP-bound heterotrimer (Gαβγ) is reformed and capable once again to interact with activated receptor.
Based on this cycle of GTP exchange and hydrolysis, the duration of heterotrimeric G-protein signaling is thought to be controlled by the lifetime of the Gα subunit in its GTP-bound state. It is precisely this interaction and the lifetime of the Gα-GTP complex which controls the extent and duration of the signal induced. If a pharmaceutical effector is to exert its most favorable response, optimization of the lifetime of signal transduction would be paramount. There is benefit in having the ability to control and possibly extend the lifetime of the Gα-GTP complex, and in doing so, the duration of the signaling response. The invention described here below in the detailed description section of the invention was designed to address this need.
In 1996, Dr. Siderovski's group [Siderovski et al. Curr Biol, 1996. 6(2): p. 211-2], along with other laboratories [Dohlman et al. Mol Cell Biol, 1996. 16(9): p. 5194-209; and Druey et al. Nature, 1996. 379(6567): p. 742-6] independently identified a superfamily of RGS (“regulator of G-protein signaling”) proteins that bind Gα subunits via a ˜120 amino-acid RGS domain and dramatically accelerate their GTPase activity (GAP activity) [Hunt et al. Nature, 1996. 383(6596): p. 175-7; and Watson et al. Nature, 1996. 383(6596): p. 172-5], thereby attenuating heterotrimer-linked signaling (FIG. 1). The discovery of RGS proteins and their GAP activity towards Gα subunits resolved apparent timing paradoxes between observed rapid physiological responses mediated in vivo by GPCRs and the slow hydrolysis activity of the cognate G-proteins seen in vitro [Arshaysky et al. Neuron, 1998. 20(1): p. 11-4]. Thus, in this capacity as negative regulators of GPCR signal transduction, the RGS proteins present themselves as excellent potential drug discovery targets [Neubig et al. Nat Rev Drug Discov, 2002. 1(3): p. 187-97], given that pharmacological inhibition of RGS domain GAP activity should lead to prolonged signaling from G-proteins activated by agonist-bound GPCRs.
There are at least 37 RGS proteins encoded by the human genome that contain the signature RGS domain (reviewed in [Neubig et al. Nat Rev Drug Discov, 2002. 1(3): p. 187-97; Siderovski et al. Crit Rev Biochem Mol Biol, 1999. 34(4): p. 215-51; and Ross et al. Annu Rev Biochem, 2000. 69: p. 795-827]). The RGS containing proteins are listed in Table 1, along with the sequences of their respective RGS domains. These proteins are grouped according to sequence homology between their RGS domains and fall into subfamilies with similar multi-domain architectures and similar target Gα subunits. For example, the GAP activity of R7 subfamily members is specific to Gαi/o. subunits [Hooks et al. J Biol Chem, 2003. 278(12): p. 10087-93], whereas that of the GEF subfamily appears specific for Gα12/13 subunits ([Suzuki et al. Proc Natl Acad Sci USA, 2003. 100(2): p. 733-8; and Kozasa et al. Science, 1998. 280(5372): p. 2109-11]; cf. [Booden et al. Mol Cell Biol, 2002. 22(12): p. 4053-61]). Based on structural and biochemical studies with RGS4, RGS domains are thought to exert their GAP activity by stabilizing a conformation of Gα that favors the transition state for GTP hydrolysis [Tesmer et al. Cell, 1997. 89(2): p. 251-61]. Several key questions are currently being addressed in the field to validate RGS proteins as bona fide drug discovery targets, including whether RGS proteins have significant roles in vivo in the physiological timing of GPCR signal transduction. There has been focus on identifying the particular function of RGS proteins in neuronal signaling in the CNS [Neubig et al. Nat Rev Drug Discov, 2002. 1(3): p. 187-97].
TABLE 1PROTEINACCESSIONNAME#RGS DOMAIN SEQUENCERGS1Q08116SLEKLLANQTGQNVFGSFLKSEFSEENIEFWLACEDYKKTESDLLPCKAEEIYKAFVHSDAAKQINIDFRTRESTAKKIKAPTPTCFDEAQKVIYTLMEKDSYPRFLKSDIYLNLL(SEQ ID NO: 21) RGS2P41220AFDELLASKYGLAAFRAFLKSEFCEENIEFWLACEDFKKTKSPQKLSSKARKIYTDFIEKEAPKEINIDFQTKTLIAQNIQEATSGCFTTAQKRVYSLMENNSYPRFLESEFYQDLC(SEQ ID NO: 22) RGS3P49796LEKLLVHKYGLAVFQAFLRTEFSEENLEFWLACEDFKKVKSQSKMASKAKKIFAEYIAIQACKEVNLDSYTREHTKDNLQSVTRGCFDLAQKRIFGLMEKDSYPRFLRSDLYLDLI(SEQ ID NO: 23) RGS4P49798SLENLISHECGLAAFKAFLKSEYSEENIDFWISCEEYKKIKSPSKLSPKAKKIYNEFISVQATKEVNLDSCTREETSRNMLEPTITCFDEAQKKIFNLMEKDSYRRFLKSRFYLDLV(SEQ ID NO: 24) RGS5O15539LDKLLQNNYGLASFKSFLKSEFSEENLEFWIACEDYKKIKSPAKMAEKAKQIYEEFIQTEAPKEVNIDHFTKDITMKNLVEPSLSSFDMAQKRIHALMEKDSLPRFVRSEFYQELI(SEQ ID NO: 25) RGS6P49758SFDEILKDQVGRDQFLRFLESEFSSENLRFWLAVQDLKKQPLQDVAKRVEEIWQEFLAPGAPSAINLDSHSYEITSQNVKDGGRYTFEDAQEHIYKLMKSDSYARFLASNAYQDLL(SEQ ID NO: 26) RGS7P49802GMDEALKDPVGREQFLKFLESEFSSENLRFWLAVEDLKKRPIKEVPSRVQEIWQEFLAPGAPSAINLDSKSYDKTTQNVKEPGRYTFEDAQEHIYKLMKSDSYPRFIRSSAYQELL(SEQ ID NO: 27) RGS8P57771SFDVLLSHKYGVAAFRAFLKTEFSEENLEFWLACEEFKKTRSTAKLVSKAHRIFEEFVDVQAPREVNIDFQTREATRKNLQEPSLTCFDQAQGKVHSLMEKDSYPRFLRSKMYLDLL(SEQ ID NO: 28) RGS9O75916NFSELIRDPKGRQSFQYFLKKEFSGENLGFWEACEDLKYGDQSKVKEKAEEIYKLFLAPGARRWINIDGKTMDITVKGLKHPHRYVLDAAQTHIYMLMKKDSYARYLKSPIYKDML(SEQ ID NO: 29) RGS10O43665SLENLLEDPEGVKRFREFLKKEFSEENVLFWLACEDFKKMQDKTQMQEKAKEIYMTFLSSKASSQVNVEGQSRLNEKILEEPHPLMFQKLQDQIFNLMKYDSYSRFLKSDLFLKHK(SEQ ID NO: 30) RGS11O94810SFRELLEDPVGRAHFMDFLGKEFSGENLSFWEACEELRYGAQAQVPTLVDAVYEQFLAPGAAHWVNIDSRTMEQTLEGLRQPHRYVLDDAQLHIYMLMKKDSYPRFLKSDMYKALL(SEQ ID NO: 31) RGS12O14924SFERLLQDPVGVRYFSDFLRKEFSEENILFWQACEYFNHVPAHDKKELSYRAREIFSKFLCSKATTPVNIDSQAQLADDVLRAPHPDMFKEQQLQIFNLMKFDSYTRFLKSPLYQECI(SEQ ID NO: 32) RGS13O14921SFENLMATKYGPVVYAAYLKMEHSDENIQFWMACETYKKIASRWSRISRAKKLYKIYIQPQSPREINIDSSTRETIIRNIQEPTETCFEEAQKIVYMHMERDSYPRFLKSEMYQKLL(SEQ ID NO: 33) RGS14O43566SFERLLQDPLGLAYFTEFLKKEFSAENVTFWKACERFQQIPASDTQQLAQEARNIYQEFLSSQALSPVNIDRQAWLGEEVLAEPRPDMFRAQQLQIFNLMKFDSYARFVKSPLYRECL(SEQ ID NO: 34) RGS16O15492SFDLLLSSKNGVAAFHAFLKTEFSEENLEFWLACEEFKKIRSATKLASRAHQIFEEFICSEAPKEVNIDHETHELTRMNLQTATATCFDAAQGKTRTLMEKDSYPRFLKSPAYRDLA(SEQ ID NO: 35) RGS17Q9UGC6NFDKMMKAPAGRNLFREFLRTEYSEENLLFWLACEDLKKEQNKKVIEEKARMIYEDYISILSPKEVSLDSRVREVINRNLLDPNPHMYEDAQLQIYTLMHRDSFPRFLNSQIYKSFV(SEQ ID NO: 36) RGS18Q9NS28SFDKLLSHRDGLEAFTRFLKTEFSEENIEFWIACEDFKKSKGPQQIHLKAKAIYEKFIQTDAPKEVNLDFHTKEVITNSITQPTLHSFDAAQSRVYQLMEQDSYTRFLKSDIYLDLM(SEQ ID NO: 37) RGS19P49795SFDKLMHSPAGRSVFRAFLRTEYSEENMLFWLACEELKAEANQHVVDEKARLIYEDYVSILSPKEVSLDSRVREGINKKMQEPSAHTFDDAQLQIYTLMHRDSYPRFLSSPTYRALL(SEQ ID NO: 38) RGS20O76081SFDKLMVTPAGRNAFREFLRTEFSEENMLFWMACEELKKEANKNIIEEKARIIYEDYISILSPKEVSLDSRVREVINRNMVEPSQHIFDDAQLQIYTLMHRDSYPRFMNSAVYKDLL(SEQ ID NO: 39) RGS21Q2M5E4NMDTLLANQAGLDAFRIFLKSEFSEENVEFWLACEDFKKTKNADKIASKAKMIYSEFIEADAPKEINIDFGTRDLISKNIAEPTLKCFDEAQKLIYCLMAKDSFPRFLKSEIYKKLV(SEQ ID NO: 40) RGS22Q9BYZ4CEHSGNKLWKDSVYFWFDLQAYHQLFYQETLQPFKVCKQAQYLFATYVAPSATLDIGLQQEKKKEIYMKIQPPFEDLFDTAEEYILLLLLEPWTKMVKSD(SEQ ID NO: 41) (2 RGSKFSDLLNNKLEFEHFRQFLETHSSSRILCAdomainsDRHWSSSGEITYRDRNQRKAKSIYIKNKYLwithinNKKYFFGPNSPASLYQQNQVMHLSGGWGKIthe RGS22LHEQLDAPVLVEIQKHVQNRLENVWLPLFLprotein)ASEQF(SEQ ID NO: 42) GRK1Q15835EFESVCLEQPIGKKLFQQFLQSAEKHLPALELWKDIEDYDTADNDLQPQKAQTILAQYLDPQAKLFCSFLDEGIVAKFKEGPVEIQDGLFQPLLQATLAHLGQAPFQEYLGSLYFLRFL(SEQ ID NO: 43) GRK2P25098TFEKIFSQKLGYLLFRDFCLNHLEEARPLVEFYEEIKKYEKLETEEERVARSREIFDSYIMKELLACSHPFSKSATEHVQGHLGKKQVPPDLFQPYIEEICQNLRGDVFQKFIESDKFTRFC(SEQ ID NO: 44) GRK3P35626TFDKIFNQKIGFLLFKDFCLNEINEAVPQVKFYEEIKEYEKLDNEEDRLCRSRQIYDAYIMKELLSCSHPFSKQAVEHVQSHLSKKQVTSTLFQPYIEEICESLRGDIFQKFMESDKFTRFC(SEQ ID NO: 45) GRK4P32298DYSSLCDKQPIGRRLFRQFCDTKPTLKRHIEFLDAVAEYEVADDEDRSDCGLSILDRFFNDKLAAPLPEIPPDVVTECRLGLKEENPSKKAFEECTRVAHNYLRGEPFEEYQESSYFSQFL(SEQ ID NO: 46) GRK5P34947DYCSLCDKQPIGRLLFRQFCETRPGLECYIQFLDSVAEYEVTPDEKLGEKGKEIMTKYLTPKSPVFIAQVGQDLVSQTEEKLLQKPCKELFSACAQSVHEYLRGEPFHEYLDSMFFDRFL(SEQ ID NO: 47) GRK6P43250DYHSLCERQPIGRLLFREFCATRPELSRCVAFLDGVAEYEVTPDDKRKACGRQLTQNFLSHTGPDLIPEVPRQLVTNCTQRLEQGPCKDLFQELTRLTHEYLSVAPFADYLDSIYFNRFL(SEQ ID NO: 48) GRK7Q8WTQ7NEHSLCEQQPIGRRLFRDFLATVPTFRKAATFLEDVQNWELAEEGPTKDSALQGLVATCASAPAPGNPQPFLSQAVATKCQAATTEEERVAAVTLAKAEAMAFLQEQPFKDFVTSAFYDKFL(SEQ ID NO: 49) SNX13Q9Y5W8PLDSILVDNVALQFFMDYMQQTGGQAHLFFWMTVEGYRVTAQQQLEVLLSRQRDGKHQTNQTKGLLRAAAVGIYEQYLSEKASPRVTVDDYLVAKLADTLNHEDPTPEIFDDIQRKVYELMLRDERFYPSFRQNALYVRML(SEQ ID NO: 50) SNX14Q9Y5W7SPLVPFLQKFAEPRNKKPSVLKLELKQIREQQDLLFRFMNFLKQEGAVHVLQFCLTVEEFNDRILRPELSNDEMLSLHEELQKIYKTYCLDESIDKIRFDPFIVEEIQRIAEGPYIDVVKLQTMRCLFEAYEHVLSLLENVFTPMFCHSDEYFRQLLRGAESP(SEQ ID NO: 51) SNX25Q9H3E2QFEDILANTFYREHEGMYMERMDKRALISFWESVEHLKNANKNEIPQLVGEIYQNFFVESKEISVEKSLYKEIQQCLVGNKGIEVFYKIQEDVYETLKDRYYPSFIVSDLYEKLL(SEQ ID NO: 52) AxinO15169SLHSLLDDQDGISLFRTFLKQEGCADLLDFWFACTGFRKLEPCDSNEEKRLKLARAIYRKYILDNNGIVSRQTKPATKSFIKGCIMKQLIDPAMFDQAQTEIQATMEENTYPSFLKSDIYLEYT(SEQ ID NO: 53 Axin2Q9Y2T1SLHSLLGDQDGAYLFRTFLEREKCVDTLDFWFACNGFRQMNLKDTKTLRVAKAIYKRYIENNSIVSKQLKPATKTYIRDGIKKQQIDSIMFDQAQTEIQSVMEENAYQMFLTSDIYLEYV(SEQ ID NO: 54) D-AKAP2O43572TLEQVLHDTIVLPYFIQFMELRRMEHLVKFWLEAESFHSTTWSRIRAHSLNTMKQSSLAEPVSPSKKHETTASFLTDSLDKRLEDSGSAQLFMTHSEGIDLNNRTNSTQNHLLLSQECDSAHSLRLEMARAGTHQVSMETQESSSTLTVASRNSPASPLKELSGKLMKSIEQDAVNTFTKYISPDAAKPIPITEAMRNDIIARICGEDGQVDP(SEQ ID NO. 55) (2 RGSYLADILFCESALFYFSEYMEKEDAVNILQFdomainsWLAADNFQSQLAAKKGQYDGQEAQNDAMILwithinYDKYFSLQATHPLGFDDVVRLEIESNICREthe D-GGPLPNCFTTPLRQAWTTMEKVFLPGFLSSAKAP2NLYYKYLprotein)(SEQ ID NO: 56) p115Q92888NSQFQSLEQVKRRPAHLMALLQHVALQFEPRhoGEFGPLLCCLHADMLGSLGPKEAKKAFLDFYHSFLEKTAVLRVPVPPNVAFELDRTRADLISEDVQRREVQEVVQSQQVAVGRQLEDFRSKRLMGMTPWEQELAQLEAWVGRDRASYEAREHRVAERLLMHLEEMQHTISTDEEKSAAVVNAIGLYMRHLGVRTKSGDKKSGRNFFRKKVMGN(SEQ ID NO: 57) PDZO15085DLEKLKSRPAHLGVFLRYIFSQADPSPLLFRhoGEFYLCAEVYQQASPKDSRSLGKDIWNIFLEKNAPLRVKIPEMLQAEIDSRLRNSEDARGVLCEAQEAAMPEIQEQIHDYRTKRTLGLGSLYG(SEQ ID NO: 58) LARGQ9NZN5CSCFQSIELLKSRPAHLAVFLHHVVSQFDPATLLCYLYSDLYKHTNSKETRRIFLEFHQFFLDRSAHLKVSVPDEMSADLEKRRPELIPEDLHRHYIQTMQERVHPEVQRHLEDFRQKRSMGLTLAESELTKLDAERDKDRLTLEKERTCAEQIVAKIEEVLMTAQAVEEDKSSTMQYVILMYMKHLGVKVKEPRNLEHKRGRIGFLPKI(SEQ ID NO: 59)
A link between pharmacological modulators of RGS functionality and signal transduction through GPCR activity could result in a drug with important clinical significance, particularly in the field of neurological disorders. It seems likely that pharmacological interventions for many neurological disorders would involve a combination of effects. Such effects may include an agonist to induce a response mediated by a GPCR complex and an inhibitor of RGS activity to prolong the effect seen with the initial agonist. However, efforts to screen compound libraries for inhibitors or activators of RGS proteins have been hampered because the GTPase activity of isolated Gα proteins is limited by GDP dissociation, so steady state GTPase activity cannot be used to measure GAP activity.
RGS proteins accelerate the rate of Gα-catalyzed GTP hydrolysis by as much as 100-fold, which provides the basis for an in vitro screening assay; moreover both types of proteins are soluble and relatively easy to produce. However, in the absence of GPCR-mediated nucleotide exchange, it is GDP release (rather than GTP hydrolysis) that is the rate-limiting step in the Gα nucleotide cycle. Thus, to examine the effect of an RGS protein in accelerating GTP hydrolysis by an isolated Gα subunit in vitro, a single round of hydrolysis of radiolabelled GTP is usually performed (a.k.a. the “single-turnover GTPase assay”). This standard assay for measuring RGS domain-mediated GAP activity is low-throughput and requires discrete steps of [γ-32P]GTP loading onto Gα, purification of the [γ-32P]GTP-Gα complex, and its immediate use before significant hydrolysis by intrinsic Gα GTPase can occur. The assay also involves isolation (in discrete time intervals) of released [32P]phosphate with activated charcoal precipitation and centrifugation, and finally scintillation counting. This type of protocol would be very difficult to incorporate into an automated high through put screening (HTS) environment. Moreover, measurement of steady state enzyme activity is the standard approach used for both basic research and HTS; all of the assumptions of Michaelis-Menten kinetic analysis are based on steady state measurements. Use of a single turnover assay thus adds additional complications in data analysis.
Reliance on reconstituted GPCR/G protein complexes and phosphate detection make steady state Gα GTPase methods unsuitable for HTS. Steady-state GTPase measurements of RGS protein GAP activity are carried out in the presence of an agonist-activated GPCR/heterotrimer complex to effect the exchange of GTP for bound GDP (see FIG. 2). This entails the use of native or heterologously co-expressed GPCRs and Gβγ proteins within membrane preparations from mammalian or Sf9 insect cells, or elaborate reconstitution of purified receptor and heterotrimer in lipid vesicles. [γ-32P]GTP radioassays utilizing charcoal to adsorb unhydrolyzed [γ-32P]GTP are generally used as a detection method, similar to the single turnover assays. The complexity and expense of using reconstituted GPCRs combined with the regulatory and disposal costs for radioactive waste limits the utility of these assay methods for an industrial HTS environment. Alternatives to radioassays have been developed that rely on colorimetric or fluorescent phosphate detection methods, however the high background levels of phosphate in biological reagents impose stringent requirements on their use. Moreover, the intent in a biochemical HTS assay is to identify inhibitors of a specific molecular target. The difficulty of deconvoluting hits from such a complex assay make it very unattractive; one might as well use a cellular assay, where the potential for interaction with multiple targets—including the GPCR itself—is not generally perceived as a disadvantage.
The present invention enables the use of biochemical assay methods to screen for modulators of RGS GAP catalytic activity. Altering the relative rates of Gα protein GTPase and GDP dissociation through mutation, so that GDP dissociation is no longer rate limiting, allows the use of steady state enzymatic assays for monitoring changes in Gα GTPase activity. As background, there is literature relating to single amino acid substitutions of important functional residues, which are highly conserved within all Gα proteins subfamilies. These mutations are identified below.
Single Mutation of a Conserved Arginine:
There are two examples of mutant Gα proteins from different subfamilies where GTP hydrolysis has been reduced more than 100-fold without disrupting RGS interactions. Native Gαi1 and Gαq have similar basal GTP hydrolysis rates (single turnover; Table 3.) of 3.0 min−1 and 0.7 min−1, respectively [Krumins et al. Methods Enzymol, 2002. 344: p. 673-85]. Mutation of a highly conserved active site Arg residue in either protein (R178C and R183C, respectively, for Gαi1 and Gαq) causes an approximate 100-fold reduction in GTPase turnover rate, but it does not abolish their functional interaction with RGS proteins. RGS4 stimulates the GTPase activity of both mutant proteins approximately 100-fold [Berman et al. Cell, 1996. 86(3): p. 445-5; and Chidiac et al. J Biol Chem, 1999. 274(28): p. 19639-43]—a factor equal to or greater than its GAP effect on the wild type protein. In the case of the Gαq R183C protein, functional interaction (i.e., stimulation of GTPase) has been demonstrated with several additional RGS proteins including RGS1, RGS2, RGS3, RGS-GAIP and with phospholipase Cβ1 [Chidiac et al. Methods Enzymol, 2002. 344: p. 686-702]. Mutation or covalent modification of the cognate Arg in three additional Gα proteins, Gαi2, Gαs and Gαt, causes similar losses of GTP hydrolysis activity [Berman et al. Cell, 1996. 86(3): p. 445-5; Freissmuth et al. J Biol Chem, 1989. 264(36): p. 21907-14; and Nishina et al. J Biochem (Tokyo), 1995. 118(5): p. 1083-9], though their interaction with RGS proteins has not yet been examined. The 20 Gα family members (i.e., reference native Gα proteins) and the locations of the critical Arginine and Alanine amino acids are presented in Table 2.
TABLE 2SEQ IDGα FamilyGenBankConserved ArgConserved AlaNO:MemberAccession #Amino Acid #Amino Acid #1i1P630961783262i2NP_0020611793273i3AAM12621178326412NP_031379205353513NP_0065632003496qNP_0020631833317sP630922013668zNP_0020641783279i/oNP_62007317932610q11NP_00205818333111q15NP_0020591863461214AAH2788617932713ONP_06626817932614oBAAM1260917932615oAAAM1260817932616olfAAM1260718835317kAAA3589617832618s2AAA5314720236719s3AAA5314818635120s4AAA53149187352
The effects of catalytic site Arg mutations on Gα GTPase activity, GDP dissociation and RGS interactions are described in Table 3.
TABLE 3GαWTWTArg MutantArg MutantProteinkcat GTPasekoff GDPkcat GTPasekoff GDPRGS InteractionsGαi13.00.087R178C 0.02-0.04>0.087RGS4Gαi24.00.02-0.04R179C 0.01-0.050.01-0.04NAGαq0.7*NAR183C 0.005NARGS1, 2, 3, 4, GAIP,PLCβ1Gαs43.80.14 R187A 0.030.27 min−1NAAll rates are per minute. All kcat values were determined using single turnover GTP hydrolysis assays with isolated Gα proteins except WT Gαq kcat, which was determined in reconstituted GPCR/Gβγ system.Data from [Posner et al., 1998] and [Coleman et al. Science, 1994. 265(5177): p. 1405-12] (Gαi1), [Nishina et al., 1995] (Gαi2), [Chidiac et al., 1999] (Gαq) and [Freissmuth et al., 1989] (Gαs4).NA = Not Available.
Single Mutation of a Conserved Alanine:
There are also several examples of Gα mutations that increase GDP dissociation without affecting GTP hydrolysis. The most striking is the A326S mutant of Gαi1, which exhibits a 25-fold increase in koff (GDP) relative to wildtype protein and an identical kcat GTP [Posner et al. J Biol Chem, 1998. 273(34): p. 21752-8]. Moreover, RGS4 stimulated the steady state GTPase activity of Gαi1 A3265 appreciably, from 1.3 min−1 to 2.2 min−1. Thus, an additional mutation that caused a relatively small decrease in kcat GTPase for the Gαi1 A326S mutant would produce a koff (GDP)/kcat (GTPase) of five or more, enabling detection of RGS GAP activity with good signal-to-noise.
Efforts to produce mutant Gα proteins have yielded proteins with decreases in their rates of GTP hydrolysis or increases in GDP dissociation from Gα proteins. Neither of these strategies have facilitated a useful system for compound library screening, where the dissociation of GDP is no longer rate limiting. Applicants envision that the ability to possibly achieve such an increase in GDP dissociation relative to GTP hydrolysis is highly likely to enable detection of RGS protein GAP activity using steady state GTPase assays. Accordingly, having the tools to examine drug interactions on RGS proteins would result in a significant improvement to the currently existing technology and potentially to important drug discoveries for the treatment of a wide variety of human disorders.